The present invention relates to a novel class of proteins for the control of plant pests. Plant pests are a major factor in the loss of the world""s commercially important agricultural crops resulting both in economic hardship to farmers and nutritional deprivation for local populations in many parts of the world. Broad spectrum chemical pesticides have been used extensively to control or eradicate pests of agricultural importance. There is, however, substantial interest in developing effective alternative pesticides.
Control of various pests through the use of biological molecules has been possible in only a limited number of cases. The best known examples of biological molecules with pesticidal uses are the xcex4-endotoxins from Bacillus thuringiensis (Bt), which is a gram-positive spore forming microorganism. Varieties of Bt are known that produce more than 25 different but related xcex4-endotoxins. Bt strains produce xcex4-endotoxins during sporulation the use of which is limited because they are active against only a very few of the many insect pests.
The limited specificity of the Bt endotoxins is dependent, at least in part, on both the activation of the toxin in the insect gut (Haider, M. Z et al., 1986, Eur. J. Biochem. 156:531-540) and its ability to bind to specific receptors present on the insects midgut epithelial cells (Hofmann, C. P. et al., 1988, PNAS 85.7844-7848). Therefore, the ability to control a specific insect pest using xcex4-endotoxins at present depends on the ability to find an appropriate xcex4-endotoxin with the desired range of activity. In many cases, no such xcex4-endotoxin is known, and It is not certain that one even exists.
Plants also routinely become infected by fungi and bacteria, and many microbial species have evolved to utilize the different niches provided by the growing plant. In addition to infection by fungi and bacteria, many plant diseases are caused by nematodes which are soil-borne and infect roots, typically causing serious damage when the same crop species is cultivated for successive years on the same area of ground.
The severity of the destructive process of disease depends on the aggressiveness of the phytopathogen and the response of the host, and one aim of most plant breeding programs is to increase the resistance of host plants to disease. Novel gene sources and combinations developed for resistance to disease have typically only had a limited period of successful use in many crop-pathogen systems due to the rapid evolution of phytopathogens to overcome resistance genes.
It is apparent, therefore, that scientists must constantly be in search of new methods with which to protect crops against plant pests. It has been found in the present invention a novel class of proteins which can be used to control plant pests.
Programmed cell death is a process whereby developmental or environmental stimuli activate a genetic program that culminate in the death of the cell (Jacobson, M. D. et al., 1997, Cell 8B: 347-354). This genetic potential exists in most, if not all, multicellular organisms. In the case of invertebrates, programmed cell death appears to play a dual role by being an integral part of both the insect development process and a response mechanism to infections particularly of viral nature (Clem, R. J. et al., 1991, Science 254: 1388-1390). Programmed cell death appears to be executed in several different manners leading to either apoptosis, atrophy or differentiation. Apoptosis is one of the best characterized types of programmed cell death encompassing cytological changes including membrane-bound apoptotic bodies and cytoplasmic blebbing as well as molecular changes such as endonucleolysis typified by the generation of oligosomal length fragments (Vaux, D. L and Strasser, A., 1996, PNAS 93:2239-2244). Although the overall apoptotic phenomenology is rather conserved among the different organisms, It is interesting to point out that, for many insect cells, cytoplasmic vacuolization and swelling rather than condensation seem to be the cytological features associated with apoptotic processes (Bowen, I. D., et al., 1996, Micros. Res. Techniq.34:202-217). The novel class of proteins disclosed within the present invention are shown to induce programmed cell death and exert a pesticidal effect.
The present invention is drawn to VIP3A(c) proteins including homologues thereof. Also provided by the invention are domains of proteins of the VIP3 class, including the toxic domain and the stabilizing domain. A preferred embodiment of the invention is the toxic domain of the VIP3A(a) protein and homologues thereof. Another preferred embodiment are antibodies to proteins of the VIP3 class, but preferably to the VIP3A(c) protein.
The invention also provides hybrid toxins comprising a toxic domain of a protein of the VIP3 class. In a preferred embodiment, the hybrid toxin is a chimeric proteins having a toxic core domain operably linked to a heterologous stabilizing domain. In another preferred embodiment, the hybrid toxin comprises an antibody, or immunologically-active fragment thereof, which immunologically recognizes the VIP3 receptor operably linked to a toxic domain from other proteins, wherein the toxin domain is obtained from a number of cytotoxic proteins including but not limited to Bacilus toxins, including endotoxins and vegetative insecticidal proteins.
Also encompassed by the invention are plants comprising a DNA sequence which encodes a protein of the VIP3 class, but preferably a VIP3A(c) protein. Preferred embodiments include plants selected from the group consisting of maize, sorghum, wheat, sunflower, tomato, cole crops, cotton, rice, soybean, sugarbeet, sugarcane, tobacco, barley, and oilseed rape. In a particularly preferred embodiment, the plant is a maize plant.
The invention also provides microorganisms comprising a heterologous DNA sequence which encodes a protein of the VIP3 class, but preferably a VIP3A(c) protein. In a preferred embodiment, the microorganism is selected from the group consisting of bacteria, baculovirus, algae and fungi. In another preferred embodiment, the microorganism is selected from the group consisting of Bacillus, Pseudomonas, Clavibacter, and Rhizobium. Further encompassed by the invention are entomocidal compositions comprising microorganisms comprising a heterologous DNA sequence which encodes a protein of the VIP3 class, but preferably a VIP3A(c) protein.
The invention further relates to plants and microorganisms further comprising a second DNA sequence which encodes a second insecticidal protein. Particularly preferred second DNA sequences are those which encode a xcex4-endotoxin, those which encode another protein of the VIP3 class, or those which encode a protein of the VIP1 or VIP2 classes. In a more preferred embodiment, the xcex4-endotoxin is active against an insect selected from the group consisting of Lepidoptera and Coleoptera. In a more particularly preferred embodiment the xcex4-endotoxin is active against Ostrinia, or Diabrotica. In another particularly preferred is a second DNA sequence which encodes a xcex4-endotoxin protein selected from the group consisting of Cry1, Cry3, Cry5 and Cry9. In a more particularly preferred embodiment, the xcex4-endotoxin is selected from the group consisting of Cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C, Cry1D, Cry1Ea, Cry1Fa, Cry3A, Cry9A, Cry9C and Cry9B. Most particularly preferred are Sendotoxins selected from the group consisting of Cry1Ab, Cry1Ba and Cry9C proteins.
The invention further provides a method of controlling insects by contacting the insects with an insecticidal amount of a protein of the VIP3 class, but preferably a VIP3A(c) protein, or an insecticidal amount of a chemical ligand to a receptor of the VIP3 class of proteins. In one preferred embodiment, the insects are contacted with a transgenic plant comprising a DNA sequence which expresses a protein of the VIP3 class, but preferably a VIP3A(c) protein in another preferred embodiment, the insects are contacted with a an entomocidal composition comprising a protein of the VIP3 class, but preferably a VIP3A(c) protein, or comprising a DNA sequence which expresses a protein of the VIP3 class, but preferably a VIP3A(c) protein. In another preferred embodiment, the transgenic plant comprises a DNA sequence which expresses the VIP3A(a) protein. In another preferred embodiment the insect is selected from the group consisting of Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthroptera, Thysanoptera, Dernaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, and Acari. In a particularly preferred embodiment, the insect is a Coleoptera or Lepidoptera. In another particularly preferred embodiment, the insect is selected from the group consisting of black cutworm (Agrotis ipsilon), fall armyworm (Spodoptera frugiperda), beet armyworm (S. exigua), yellow striped armyworm (S. omithogalli), southwestern corn borer (Diatraea grandiosella), sugarcane borer (D. saccharalis), corn earworm (Helicoverpa zea), Mediterranean corn borer (Sesamia nonagroides), cabbage looper (Trichoplusia ni), velvetbean caterpillar (Anticarsia gemmatalis), diamondback moth (Plutella xylostella) and tobacco budworm (Heliothis virescens).
Also provided by the invention is a method of controlling insects wherein the transgenic plant or microorganism further comprises a second DNA sequence which encodes a second insecticidal protein such as those mentioned hereinbefore.
The invention further provides recombinant DNA sequences which encode a VIP3A(c) protein including homologues thereof. In another preferred embodiment, the DNA sequence is a synthetic sequence which has been altered for optimum expression in a plant, particularly where the DNA sequence has been optimized for expression in a maize plant. Also preferred are DNA sequences which comprise both a synthetic portion and a native portion. In a particularly preferred embodiment, the DNA sequence encoding the VIP3A(c) protein has been optimized for expression in a maize plant. Another preferred embodiment are DNA sequences which are homologous to a DNA sequence which encodes a VIP3A(c) protein. Particularly preferred are DNA sequences which hybridize under moderately stringent conditions to the vip3A(c) coding sequence. Yet another embodiment of the invention is a recombinant DNA sequence which expresses a protein of the VIP3 class, preferably a VIP3A(c) protein, under the control of a heterologous promoter, or wherein the coding regions is incorporated into the genome of an organism where it is not naturally expressed or is expressed at higher levels than that occuring naturally.
The invention is further drawn to a method of identifying and isolating homologues of a VIP3A(c) protein. or of a DNA sequence which encodes said protein.
Also provided by the invention are expression cassettes comprising a promoter operably linked to a DNA sequence encoding a protein of the VIP3 class, but preferably a VIP3A(c) protein. In one preferred embodiment the promoter is selected from the group consisting of constitutive, tissue-preferred and tissue-specific promoters for expression in plants. In a particularly preferred embodiment, the promoter is selected from the group consisting of the ubiquitin, PEP carboxylase, LPT and MTL promoters. In another preferred embodiment, the promoter is functional in a microorganism.
The invention further provides a receptor to a protein of the VIP3 class and DNA sequences which. In one embodiment of the invention, the receptor comprises a death domain and a repeated EGF-motif. A more preferred embodiment of the invention comprises a receptor to the VIP3A(a). A more particularly preferred embodiment is the receptor protein sequence set forth in SEQ ID NO:9, and homologues thereto. Also encompassed by the invention are DNA sequences which encode these receptor proteins, e.g., the DNA sequence set forth in SEQ ID NO:8 and homologues thereto. The cDMA for the VIP3 receptor is contained in plasmid pCIB7113, which was deposited under the Budapest Treaty with the NRRL [Agricultural Research Service, Patent Culture Collection (NRRL), Northern Regional Research Center, 1815 North University Street, Peoria, Ill. 61604, USA] on Mar. 29, 1997 and has accession number B-21676. Antibodies to a receptor of the VIP3 class of proteins are also encompassed by the invention.
Also provided by the invention is a method of identifying a compound as a VIP3 receptor chemical ligand having pesticidal activity comprising exposing a cell, preferably an insect cell, to a test compound, and assaying said cell for apoptotic activity. In another embodiment of the invention, the method comprises measuring specific binding between VIP3 receptor and a test compound. A preferred embodiment are VIP3 receptor ligands identified by the method.
xe2x80x9cPlant pestxe2x80x9d means any organism known to associate with plants and which, as a result of that association, causes a detrimental effect on the plant""s health and vigor. Plant pests include but are not limited to fungi, bacteria, insects, and nematodes. The term plant as used herein encompasses whole plants and parts of plants such as roots, stems, leaves and seed, as well as cells and tissues within the plants or plant parts.
The xe2x80x9cVIP3 class of proteinsxe2x80x9d comprises VIP3A(a), VIP3A(b) VIP3A(c) and their homologues. xe2x80x9cHomologuexe2x80x9d is used throughout to mean that the indicated protein or polypeptide bears a defined relationship to other members of the VIP3 class of proteins. This defined relationship includes but is not limited to, 1) proteins which are at least 70%, more preferably 80% and most preferably 90% identical at the sequence level to another member of the VIP3 class of proteins while also retaining pesticidal activity, 2) proteins which are cross-reactive to antibodies which immunologically recognize another member of the VIP3 class of proteins, 3) proteins which are cross-reactive with a receptor to another member of the VIP3 class of proteins and retain the ability to induce programmed cell death, and 4) proteins which are at least 70%, more preferably 80% and most preferably 90% identical at the sequence level to the toxic core region of another member of the VIP3 class of proteins while also retaining pesticidal activity.
A xe2x80x9chybrid toxin xe2x80x9d is used to indicate a genetic fusion, having domains operably linked so that, when translated, a functional chimeric protein is formed having, in the aggregate, the properties of the individual domains. xe2x80x9cDomainxe2x80x9d is used to indicate a region or portion of a protein or confers a recognizable function or structure which contributes to the overall functionality of the protein. It is recognized that a DNA sequence which encodes a protein domain is also encompassed by this definition.
xe2x80x9cHeterologousxe2x80x9d is used to indicate that a protein, polypeptide or nucleotide sequence has a different natural origin with respect to its current host. For example, if a vip3A(a) gene from a Bacillus thuringiensis is genetically transformed into a plant cell, then the gene is described as being heterologous with respect to its current host, which is the plant cell. Furthermore, if a vip3A(a) gene from Bacillus thuringiensis is genetically transformed into a Pseudomonas bacterium, then the gene is also described as being heterologous with respect to the Pseudomonas. xe2x80x9cHeterologousxe2x80x9d is also used to indicate that one or more of the domains present in a chimeric protein, polypeptide or nucleotide sequence differ in their natural origin with respect to other domains present. For example, if the toxic domain from VIP3A(a) protein is fused to the binding domain from the VIP1A(a) protein to make a functional insecticidal protein, then the chimeric fusion would have domains that are heterologous to each other. In addition, a heterologous chimeric protein or polypeptide comprising the fusion of a toxic domain from VIP3A(a) protein to the binding domain from the VIP1A(a) protein, when expressed in a plant, would also be considered heterologous with respect to the plant host.
The term xe2x80x9cchimericxe2x80x9d is used to indicate that the protein, polypeptide, or nucleotide sequence is comprised of domains at least one of which has an origin that is heterologous with respect to the other domains present. These chimeric proteins or polypeptide are encoded by chimeric nucleotide sequences which have been fused or ligated together resulting in a coding sequence which does not occur naturally. Such chimeric constructions may also be designated as xe2x80x9crecombinant.xe2x80x9d
xe2x80x9cExpression cassettexe2x80x9d as used herein means a DNA sequence capable of directing expression of a gene in plant cells, comprising a promoter operably linked to an amino acid coding region which is operably linked to a termination region. The gene may be chimeric, meaning that at least one component of the gene is heterologous with respect to at least one other component of the gene. The gene may also be naturally occurring, but which has been obtained in a recombinant form useful for genetic transformation of a plant or microorganism.